Volume 75, Issue 1, Pages 528-537 (July 1998) Time-Resolved Analysis of Macromolecular Structures During Reactions by Stopped- Flow Electrooptics  Dietmar Porschke  Biophysical Journal  Volume 75, Issue 1, Pages 528-537 (July 1998) DOI: 10.1016/S0006-3495(98)77542-3 Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 1 Cells for stopped-flow electric field-jump measurements. (a) Quartz cell with a thin metal layer around the upper and lower plates deposited from the gas phase; first a contact layer is made from chromium, which is then used as a basis for a gold layer. (b) Quartz cell with platinum electrodes integrated inside the upper and lower plates with electrical connections to the outside by platinum wires. Biophysical Journal 1998 75, 528-537DOI: (10.1016/S0006-3495(98)77542-3) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 2 Mounting unit for the quartz cell. The inner mount is shown at the center, with windows for spectroscopic detection and with elastic contacts for electrical connections; the two parts of the outer mount (top and bottom) hold the inner mount in position for insertion into the stopped-flow instrument. Biophysical Journal 1998 75, 528-537DOI: (10.1016/S0006-3495(98)77542-3) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 3 Construction of the stopped-flow instrument: top view (left) and side view (right). Biophysical Journal 1998 75, 528-537DOI: (10.1016/S0006-3495(98)77542-3) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 4 Block diagram of the stopped-flow field-jump instrument. Biophysical Journal 1998 75, 528-537DOI: (10.1016/S0006-3495(98)77542-3) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 5 Electric dichroism transient measured at 248.2nm for a mixture of 50μMbp 95-bp fragment and 10μM ethidium in buffer A at 10°C at a time delay of the pulse 2ms after stop of the flow. The delays of the optical responses after pulse application/termination and the deviations from the single exponential form are due to convolution with the detector response function; the detector has been adjusted to a relatively slow response in order to increase the signal to noise ratio. Because the detector response function can be measured at a high accuracy, deconvolution is without problems (Porschke and Jung, 1985). The temperature jump induced by the field pulse is calculated to be 0.13°C. Biophysical Journal 1998 75, 528-537DOI: (10.1016/S0006-3495(98)77542-3) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 6 Dichroism amplitudes ΔA measured at 313.0nm for 50μMbp 95-bp fragment and 10μM ethidium in buffer A as a function of the time delay after mixing td (time delay after stop of the flow+dead time of the stopped flow, 10°C, 12.5kV/cm). Each of the experimental points represents the results obtained from an average of two stopped-flow shots, using single field pulses per shot with pulse lengths of 10μs. The line represents a least-squares fit of the data by a single exponential of 3.6ms. Biophysical Journal 1998 75, 528-537DOI: (10.1016/S0006-3495(98)77542-3) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 7 Dichroism decay times τ measured at 248.2nm for 50μMbp 95-bp fragment and 10μM ethidium in buffer A as a function of the time delay after mixing td (10°C, 12.5kV/cm). Each of the experimental points represents the results obtained from an average of two stopped-flow shots, using single field pulses per shot with pulse lengths of 10μs. The line represents a least-squares fit of the data by a single exponential of 4.0ms [τ(td=0)=1.179μs and τ(td=∞)=1.469μs]. Biophysical Journal 1998 75, 528-537DOI: (10.1016/S0006-3495(98)77542-3) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 8 Dichroism amplitudes ΔA measured at 313.0nm for 50μMbp 95-bp fragment and 10μM ethidium in buffer B as a function of the time delay after mixing td (10°C, 12.5kV/cm). Each of the experimental points represents the result from a stopped-flow shot, using a single pulse per shot with pulse lengths of 14μs. The line represents a least-squares fit of the data by exponentials of 1.1ms and 120ms. Biophysical Journal 1998 75, 528-537DOI: (10.1016/S0006-3495(98)77542-3) Copyright © 1998 The Biophysical Society Terms and Conditions